Technical Abstract:
Underground adventitious buds of leafy spurge undergo three well-defined phases of dormancy, para-, endo-, and ecodormancy, throughout the year. In this study, relationships between carbohydrate metabolism and bud dormancy were examined and real-time PCR was used to determine if shifts in carbohydrate contents correlate with the expression levels of some carbohydrate metabolism genes. Our results indicated that many carbohydrate metabolism genes were differentially-regulated after paradormancy release and in response to seasonal signals. Among these genes, a specific ß-amylase transcript increased 100-fold after growth induction and increased 16,000-fold from July to December. This ß-amylase was represented by two genes, Ee-BAM1 and Ee-BAM2. The deduced amino acid sequences of these two genes are very similar at the N-terminal end but are disparate at the C-terminal. Both contain a nearly identical, predicted 48-amino acid plastid transit peptide. Only Ee-BAM1, not Ee-BAM2, can be amplified by PCR using gene-specific primers, indicating that Ee-BAM2 is organ specific and/or not abundant. Immunoblot analyses identified a 29-kD and a 35-kD protein; the 29-kD protein could be the mature Ee-BAM1 where the transit peptide was cleaved. Unlike transcript expressions, both 35-kD and 29-kD proteins were constitutively expressed in growth-induced and seasonal samples. Immunolocalization indicated that Ee-BAM1 is in the cytosol of cells constituting leaf primordium and procambium at the tip of the bud. Ee-BAM1 also surrounds the amyloplasts in mature cells toward the base of the bud. These observations implicate that Ee-BAM1 may have dual functions; it serves as nutrient reserve in the cytosol and acts as degrading enzyme at the surface of amyloplasts.
Key words: ß-amylase, carbohydrate metabolism, dormancy, leafy spurge.